Applied Clay Science 75–76 (2013) 126–133

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Applied Clay Science

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Research paper

Thiamine pyrophosphate intercalation in layered double hydroxides

(LDHs): An active bio-hybrid catalyst for pyruvate decarboxylation
M. Baikousi a, Ag. Stamatis b, M. Louloudi b, M.A. Karakassides a,⁎
a
Department of Materials Science and Engineering, University of Ioannina, GR-45110 Ioannina, Greece
b
Department of Chemistry, University of Ioannina, GR-45110, Ioannina, Greece

a r t i c l e i n f o a b s t r a c t

Article history: The intercalation of thiamine pyrophosphate (TPP) into Zn–Al and Mg–Al layered double hydroxides has
Received 26 July 2012 been investigated using two different methods: the co-precipitation procedure (at different pH values) and
Received in revised form 7 February 2013 the ion-exchange reaction. The structure of the hybrid materials was studied by X-ray diffraction (XRD)
Accepted 11 February 2013
and Fourier transform infrared (FT-IR) spectroscopy verifying that the TPP molecules prefer creating a bilayer
Available online 14 March 2013
arrangement at pH = 7.5, 7.75 and 8, while in the case of pH = 7 a monolayer of TPP molecules coexists in the
Keywords:
interlayer space of LDHs. In addition, the thermal decomposition of the bio-hybrid materials was examined
Layered double hydroxide by thermogravimetry and differential thermal analysis (TG–DTA). The total amount of the deintercalated
Thiamine pyrophosphate TPP molecules from LDHs was calculated after de-intercalation reactions in the presence of carbonate ions
Intercalation by Ultraviolet (UV) spectroscopy. Furthermore, the hybrid materials (TPP/LDH) have been tested as biomi-
Catalysis metic catalysts for decarboxylation of pyruvic acid showing a comparable activity to the homogenous system
Pyruvate decarboxylation (TPP). Our combined data confirm that thiamine pyrophosphate can be safely loaded and stored into LDH's
layers forming a new bio-active hybrid material.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Layered double hydroxides (LDHs) or anionic clays, which are wide-
ly known as two dimensional layered compounds possessing anion ex-
change properties, have received considerable attention due to their
special intercalation properties. In nature LDHs formed from the
weathering of basalts on precipitation in saline water sources (Rives,
2001). LDHs can be also readily synthesized in the laboratory using
co-precipitation methods at varied or constant pH values. The naturally
occurring material is hydrocalcite or its hexagonal analog, manasseite
([Mg6 Al2(OH)16][CO3]·4H2O). The general composition of these mate-
rials can be represented as [MII 1−x MIIIx(OH)2]x+[(A m−)x/m n H2O]
where MII is a divalent metal cation (i.e. Mg, Ni, Cu, Zn), M III is a trivalent
metal cation (i.e. Al, Cr, Fe, V) and Am− represents an anion of charge m
such as CO32−, Cl−, SO42−, and NO3−. LDHs have been studied for their
potential use in a wide range of important areas, i.e. catalysis, photo-
chemistry, electrochemistry, polymerization, magnetization, biomedi-
cal science, and environmental applications (Cavani et al., 1991;
Costantino et al., 2009; Li and Duan, 2006; Newman and Jones, 1998;
Rives, 2001).
Recently, the use of LDHs as host materials to synthesize biomolecule/
inorganic hybrids has attracted considerable interest due to their good
biocompatibility, low cytotoxicity and their framework structure which
provides full protection for the loaded biomolecules in the interlayer.
⁎ Corresponding author. Tel.: +30 26510 07276; fax: +30 26510 07074.
E-mail address: mkarakas@cc.uoi.gr (M.A. Karakassides).
These two-dimensional materials could act as a stable host matrix for
stabilization, storage and delivery of nucleotides, amino acids (Aisawa
et al., 2001, 2003, 2004; Fudala et al., 1999a, 1999b; Nakayama et al.,
2004; Wei et al., 2007; Whilton et al., 1997; Yuan et al., 2004), vitamins
(Choy and Son, 2004; Choy et al., 2004; Gasser, 2009; Hwang et al.,
2001) and other anionic biomolecules (Aisawa et al., 2005; Choy et al.,
1999, 2000, 2001; Costantino et al., 2008; Kwak et al., 2002; Lotsch et
al., 2001). The guest biomolecules can be easily recovered through sim-
ple deintercalation reactions without the change of the host compound.
There are various ways to introduce the anionic substances into the
interlayer space of LDHs (Cavani et al., 1991; Rives, 2001). Generally,
the most used methods are i) the in-situ co-precipitation method in
which the synthesis of LDH occurs in the presence of the desirable an-
ionic species and ii) anionic exchange in which a LDH precursor with
NO3− or Cl− as the counter-anion is exchanged by the anionic species
to form the LDH-organic hybrids.
Thiamine pyrophosphate (TPP), the biologically active derivative
of thiamine which is a water-soluble B-complex vitamin (vitamin
B1), serves as a cofactor in a number of enzymatic processes found
in numerous major metabolic pathways, of all living systems. TPP
plays a key role in the metabolism of carbohydrates and the biosyn-
thesis of side chain of amino acids such as α-ketoacid (e.g. pyruvate)
decarboxylases, dehydrogenases and oxidases, transketolase, benzal-
dehyde lyase, acetolactate and deoxyxylulose 5-phosphate synthase
(Jordan, 2003; Schellenberger, 1998). From a structural point of
view TPP molecule is constituted of a pyrophosphate group and two
heterocyclic rings, a pyrimidine and a thiazole with an alkylated

0169-1317/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.clay.2013.02.006
 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133
nitrogen atom (Pletcher et al., 1977). TPP molecules occur as anions
under physiological conditions around pH 6–7 due to the deproton-
ation of the pyrophosphate group (Malandrinos et al., 1998, 2000).
Therefore, the possible electrostatic interactions of TPP with the
basal cationic layers of LDH, make the LDHs favorable host structures
for TPP guest molecules in order to develop a new biohybrid LDH
material. A convenient immobilization of thiamine pyrophosphate
on a silica surface led to a very active heterogenized biocatalyst
for pyruvate decarboxylation (Vartzouma et al., 2002). Recently,
simple thiamine without pyrophosphate chain has been intercalat-
ed into mineral clays (Joshi et al., 2009). In this context, TPP seems
to be a promising candidate for loading in the interlayer space of
LDH.
Here we present thiamine pyrophosphate intercalation into the
layers of LDHs by ion exchange and co-precipitation methods. The
structure of the new biomolecule/inorganic hybrids has been studied
by XRD and FT-IR analysis. The bio-availability and reactivity of guest
thiamine pyrophosphate molecules, have been evaluated for pyru-
vate decarboxylation whereas the TPP/LDH hybrid systems were
used as catalysts. In addition, deintercalation of loaded TPP mole-
cules from LDH layers has occurred in the presence of carbonate
anions.
2. Experimental
2.1. Materials
Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was purchased
from Sigma-Aldrich (96482), magnesium nitrate hexahydrate
(Mg(NO3)2·6H2O) from Fluka (63084), aluminum nitrate nonahydrate
(Al(NO3)3·9H2O) from Fluka (63274), thiamine pyrophosphate
(C12H19ClN4O7P2S) from Sigma-Aldrich (C8754), sodium hydroxide
(NaOH) from Merck (1.06498), methanol (CH3OH) 99,8% from Panreac
(131091) and sodium pyruvate (CH3COCOONa) from Sigma-Aldrich
(P2256).
2.2. Synthesis of NO3−/ZnAl-LDH
NO3−/ZnAl-LDH was prepared under nitrogen atmosphere by follow-
ing a standard co-precipitation procedure (Cavani et al., 1991; Miyata,
1975). To a 100 ml decarbonated aqueous solution of 0.075 mol
Zn(NO3)2·6H2O and 0.025 mol Al(NO3)3·9H2O (Zn2+/Al3+ =3/1)
aqueous NaOH solution 2.5 M was added dropwise until pH value
reached to 9. After aging with stirring at 100 °C, the white precipitate
was centrifuged and washed several times with water and finally was
dried at 80 °C for 24 h.
2.3. Intercalation of thiamine pyrophosphate for TPP/M 2+Al-LDH
(M 2+ = Zn 2+, Mg 2+)
Intercalation of thiamine pyrophosphate (TPP) for M 2+Al-LDH
was performed following two different procedures, anion exchange
method and co-precipitation method.
2.3.1. Anion exchange method
The TPP-7/ZnAl-LDH(ae) hybrid material with general formula:
[Zn2+0.75Al3+0.25(OH)2]0.25+ [C12H19N4O7P2Sn−]0.25/n. mH2O (1≤n≤3),
was synthesized by anion exchange reaction of the NO3−/ZnAl-LDH
with thiamine pyrophosphate anions at pH 7. In particular, 1 g of
NO3−/ZnAl-LDH (anion exchange capacity: 1.9 mmol/g) was dis-
persed in 150 ml of aqueous solution (decarbonated water) of 9 mmol
TPP (4.3 g thiamine pyrophosphate) and the pH value of the suspension
was adjusted to 7 by adding aqueous solution of NaOH (2.5 M). The sus-
pension was stirred at 25 °C for 36 h under nitrogen atmosphere,
centrifuged and washed several times with decarbonated water and fi-
nally was dried at room temperature for 24 h.
127
2.3.2. Co-precipitation method
The TPP-7/ZnAl-LDH, TPP-7.5/ZnAl-LDH, TPP-8/ZnAl-LDH and
TPP-7.75/MgAl-LDH hybrid materials with general formula:
[M2+0.67Al3+0.33(OH)2] 0.33+ [C12H19N4O7P2S n−]0.33/n. mH2O (where
M2+ is Zn2+ or Mg2+ and 1 ≤ n ≤ 3), were synthesized by applying
the co-precipitation method at different pH values (7, 7.5, 8
and 7.75 respectively). An aqueous solution of 0.012 mol Μ 2+(ΝΟ3)2
6H2O·(Μ 2 + = Μg 2 + or Zn 2 +) and 0.006 mol Al(NO3)3·9H2O
(Μ 2 +/Al = 2/1) was added to an aqueous solution of TPP
(TPP/Al = 1.8/1), whereas the pH value of the mixture was ad-
justed by adding NaOH (2.5 M) dropwise. The white precipitated
products were stirred at 25 °C for 5 h. Then, the solids were centrifuged
and washed several times with water and finally they were dried at
room temperature for 24 h.
2.4. Synthesis of CO32−/ZnAl-LDH as reference sample
CO32−/ZnAl-LDH was prepared by applied a standard co-precipitation
procedure (Cavani et al., 1991; Miyata, 1975). In 50 ml aqueous solution
of Zn(NO3)2·6H2O and Al(NO3)3·9H2O (Zn2+/Al3+ =3/1) an aqueous
solution of Na2CO3 (CO3/Al =1.8/1) was added. The pH value of the mix-
ture was adjusted to 9 by adding dropwise an aqueous solution of NaOH
(2.5 M). The white solids, which were precipitated immediately, were
stirred at 60 °C for 24 h in the mother solution. They were centrifuged
and washed several times with water and finally were dried at 80 °C
for 24 h.
2.5. Catalytic evaluation of hybrid materials
The hybrid materials (TPP/LDHs) have been evaluated as hetero-
geneous catalysts for pyruvate decarboxylation. All reactions were
carried out at 37 °C in methanol (1 ml) with sodium pyruvate as sub-
strate (200 μmol), loaded TPP from TPP-7/ZnAl (ae) or TPP-7/ZnAl as
catalyst (20 μmol) and NaOH (40 μmol). The ratio of [catalyst:base:
substrate] was equal to [1:2:10]. Bromobenzene was used as internal
standard. The substrate conversion was monitored by GC–MS, by re-
moving the small samples of the reaction mixture. To establish the
identity of the products unequivocally, the retention times and spec-
tral data were compared to those of commercially available com-
pounds. Blank experiments showed that without thiamine-based
catalyst there is no substrate conversion.
2.6. Deintercalation experiments
Deintercalation of TPP from TPP-7/ZnAl (ae) and TPP-7/ZnAl was
carried out by dispersing 0.01 g of the hybrid materials in 100 ml of
0.3 M Na2CO3 aqueous solution at room temperature. The mixtures
were stirred for 24 h while the pH of the suspensions was adjusted
to 2.2 by adding hydrochloric acid solution (1 M). The release amount
of TPP was determined spectrophotometrically at 245 nm (Özgür and
Koyuncu, 2002) using a calibration curve. The deintercalated mate-
rials were characterized using FT-IR spectroscopy and X-ray powder
diffraction.
The calibration curve was based on different standard 0.1 M HCl aque-
ous solutions of TPP (known concentrations 0–1.5×10−1 μmol/ml) and
found out that absorbance at 245 nm exhibits linear relationship with
the TPP concentration (absorbance=16.76× [TPP]+0.071, R2 =0.999,
linear: 0–0.12 μmol/ml).
2.7. Characterization
Infrared (FT-IR) spectra of samples in powder form, dispersed in
KBr pellets, which were the average of 32 scans at 2 cm −1 resolution,
were measured with a Perkin-Elmer GX, Fourier transform spectrom-
eter in the frequency range of 400–4000 cm −1. The band component
analysis was performed by utilizing the Origin 7.5 peak fitting
128 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133
program (PFM). We have chosen the minimum number of bands that
give a reasonable agreement between the experimental and the calculat-
ed spectra. X-ray powder diffraction data were collected on a D8 Advance
Bruker diffractometer using Cu Kα (40 kV, 40 mA, λ=1.54178 Å) radi-
ation and a secondary beam graphite monochromator. Diffraction pat-
terns were collected in the 2θ range from 1 to 80°, in steps of 0.02° and
2 s counting time per step. Thermogravimetric (TGA) and differential
thermal analysis were performed using a Perkin Elmer Pyris Dia-
mond TG/DTA instrument. Samples of approximately 5 mg were
heated in air from 25 °C to 900 °C, at a rate of 5 °C/min. UV–Visible
(UV–vis) spectra of solutions were measured in quartz vesicle with a
UV-2401(PC)-Shimadzu two-beam spectrophotometer in the range
200–400 nm at a step of 0.5 nm using a combination of deuterium
and halogen lamps as sources. GC analyses were performed using a
Shimadzu GC-17A gas chromatograph coupled with a GCMS-QP5000
mass spectrometer.
3. Results and discussion
Fig. 1 shows the powder XRD patterns of thiamine pyrophosphate
(TPP), the pristine NO3−/ZnAl-LDH (a) and hybrid materials TPP/M(II)
Al-LDH (b–f) which were prepared by different synthetic approaches
(anion-exchange and co-precipitation methods). The XRD pattern of
NO3−/ZnAl-LDH (a) exhibits the main diffraction at 2θ = 9.96 ο which
corresponds to the basal spacing d003 = 0.89 nm and gallery height
Fig. 1. XRD patterns of thiamine pyrophosphate (TPP), pristine NO3−/ZnAl-LDH (a) and hy-
brid LDHs TPP-7/ZnAl(ae) (b), TPP-7/ZnAl (c), TPP-7.5/ZnAl (d), TPP-7.75/MgAl (e) and
TPP-8/ZnAl (f).
c0 = 0.41 nm (c0 = d003 − 0.48 nm, where 0.48 nm is the thickness
of LDH layer (Rives, 2001)).
In the XRD pattern of hybrid material TPP-7/ZnAl-LDH(ae) (b), pre-
pared by the anion-exchange reaction of the NO3−/ZnAl-LDH with thia-
mine pyrophosphate at pH= 7, a shift of the 003 diffraction to lower
angles is observed, indicating the successful intercalation of TPP mole-
cules into LDH. In particular, this pattern shows a main diffraction at
2θ = 5.92° which corresponds to the basal spacing d003 = 1.5 nm and
gallery height c0 = 1.02 nm. However a second diffraction with lower
intensity, is appeared at 2θ = 3.5 o (d′003 = 2.52 nm) corresponding to
the gallery height of c′0 = 2.04 nm. The appearance of these two main
diffractions in the XRD pattern of hybrid material indicates two
rearrangements of TPP molecules between LDH layers in different crys-
tallites. Taking into account that the size of TPP is ~1.3 nm and the gal-
lery height c0 and c′0 are 1.02 and 2.04 nm respectively, a possible
model of TPP intercalation is proposed: a) a monolayer formation
(Fig. 2a and b) a TPP bilayer formation (Fig. 2b) via the stacking interac-
tion of aromatic rings of TPP between the LDH basal layers. The fact that
the d003 diffraction exhibits higher intensity and it is sharper than d′003
diffraction, indicates that the formation of TPP monolayer is the dominant
type of rearrangement for the hybrid material TPP-7/ZnAl-LDH(ae). Sim-
ilar features are observed in the XRD pattern (c) of the hybrid material
TPP-7/ZnAl-LDH synthesized by a different procedure (co-precipitation
method) at pH=7. In the XRD patterns (d–f) of the hybrid materials pre-
pared by co-precipitation method at different pH values (pH=7.5, 7.75,
8), only one main diffraction at 2θ=3.5o is observed, which corresponds
to the gallery height c′0 =2.04 nm. This distance fits well to a bilayer for-
mation of TPP between LDH layers by π–π stacking interactions of aro-
matic rings of TPP. It seems that the TPP prefer forming a bilayer
rearrangement at pH>7.5 while in hybrid materials prepared at pH=7,
the TPP monolayer coexists with the TPP bilayer in the LDHs. In addition,
the LDH crystallinity in all hybrid materials prepared by co-precipitation
method is lower attributed to the lower pH of reaction solution.
Fig. 3 displays the infrared spectra of TPP, pristine NO3−/Zn-Al LDH
and hybrid materials TPP-7/ZnAl-LDH(ae) and TPP-7/ZnAl-LDH pre-
pared by anion-exchange and co-precipitation methods, respectively.
The spectrum of pristine NO3−/ZnAl LDH shows two broad absorption
bands at 3471 and 600 cm −1 due to vibration modes of hydroxyl
groups of LDH layers and one more at 1620 cm −1 due to bending vi-
bration modes of interlayer water molecules (Kagunya et al., 1998;
Kloprogge and Frost, 1999; Rives, 2001). The very strong absorption
band at 1386 cm −1 and the weakly band at 823 cm −1, are assigned
to asymmetric stretching and symmetric deformation vibrations of
NO3−interlayer anions, respectively. In the spectrum of the hybrid ma-
terial TPP-7/ZnAl-LDH(ae), these bands are absent. Moreover the ap-
pearance of new absorption bands due to the vibration modes of
intercalated thiamine pyrophosphate anions, confirms the complete
replacement of nitrate ions by TPP molecules. The spectrum of the
pristine TPP exhibits absorption bands corresponding to vibration
modes of the pyrophosphate group and the aromatic pyrimidine
and thiazole rings (Table 1). In the spectrum of the hybrid material
TPP-7/ZnAl-LDH(ae), these bands are shifted to different frequencies
(Table 1) due to the intercalation of TPP in the interlayer space of
LDH. This gives information about the symmetry of the intercalated
molecules and their interactions with the hydroxylated LDH layers.
Also, these bands are observed at the same frequencies in the spectra
of the hybrid material TPP-7/ZnAl-LDH (Fig. 3:TPP-7/ZnAl).
As mentioned before, the molecules of TPP are negatively charged
at pH value > 6.5 (Malandrinos et al., 1998, 2000) due to the depro-
tonation of the pyrophosphate groups. Therefore, the possible elec-
trostatic interaction of TPP with the basal cationic layers of LDH
occurs via the anionic pyrophosphate groups. These interactions
have an effect on the energy of P\O, O\P\O and P_O bonds of
TPP molecules. Thus the bands assigned to the vibration modes of
these bonds are observed at different frequencies in the infrared spec-
tra of hybrid materials compared to that of the pristine TPP (Table 1)
 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133
Fig. 2. Schematic illustrations of pristine NO3−/ZnAl-LDH and hybrid materials: (a) TPP monolayer intercalation and (b) TPP bilayer layer intercalation via stacking.
indicating TPP/LDH interaction. In the spectra of TPP-7/ZnAl, the
strong absorption band at 1386 cm −1 is still observed indicating
co-intercalation of ΝΟ3− into the LDH. The presence of nitrate anions
in the interlayer region of LDH is a result of the applied synthetic pro-
cedure, where metal nitrates were used as metal sources for hybrid
formation (Kloprogge et al., 2002, 2004). Similar behavior is observed
in the infrared spectra (Fig. 5) of the hybrid materials prepared by the
co-precipitation method at different pH values (pH = 7.5, 7.75, 8).
In the infrared spectra of the hybrid materials, the low frequency
region at 450–700 cm −1 differs from the same region in the infrared
spectrum of the pristine TPP due to the presence of the vibration
modes of hydroxyl groups of LDH layers. Band component analysis,
of these regions in the infrared spectra of TPP-7/ZnAl-LDH(ae) and
TPP-7/ZnAl-LDH, reveals the presence of seven bands (Fig. 3 — right
side). In the spectra of TPP-7/ZnAl(ae) and TPP-7/ZnAl the bands at
604, 549 cm −1 and 609, 548 cm −1, respectively, correspond to hy-
droxyl translation modes which are mainly influenced by the divalent
and trivalent metal in the LDH's layer structure (Kloprogge and Frost,
1999). The bands at 520, 579 and 652 cm −1 (518, 579 and 650 cm −1
for TPP-7/ZnAl) and this at 495 cm −1 (494 cm −1 for TPP-7/ZnAl)
correspond to deformation vibrations of thiazole and pyrimidine
ring respectively of the intercalated TPP. Finally, the band at
477 cm −1 can be assigned to the bending vibration modes of P\O
bonds of the pyrophosphate groups (Leopold et al., 2005).
The %TG curves of TPP, pristine NO3−/Zn-Al LDH and TPP/LDHs are
represented in Fig. 4. In the case of TPP, two weight losses are observed
a) at 200–400 °C (weight loss 33%) and b) at 400–750 °C (weight loss
51%) due to the decomposition and combustion of TPP, respectively.
The residual mass (16%) corresponds to the phosphorous oxides from
TPP's pyrophosphate groups. In the case of pristine NO3−/Zn–Al LDH
three weight losses are observed, at 40–500 °C, attributed to the remov-
al of absorbed/interlayer water, the dehydroxylation of the LDH basal
layer and the elimination of interlayer anions (NO3−) accompanied by
129
NO2 and NO evolution (Velu et al., 1997). Here, the residual mass
(63%) corresponds to the zinc and aluminum oxides that derived from
the LDH basal layer. On the other hand, the thermal decomposition of
the TPP/LDHs is influenced by the synthetic procedure: pH value, the
nature of LDH components (Fig. 4b–4f). In all hybrids, the total weight
loss (TPP-7/ZnAl(ae): 42%, TPP-7/ZnAl: 57.6%, TPP-7.5/ZnAl: 51.3%,
TPP-7.75/MgAl: 58%, TPP-8/ZnAl: 52.1%) occurs in four steps. The first
step, which corresponds to the removal of absorbed and interlayer
water, is observed, from room temperature to ~150 °C. The second
step showed at 150–500 °C and is assigned to the dehydroxylation of
the LDH basal layer, the elimination of the co-intercalated NO3− and
the decomposition of the intercalated TPP. The combustion of the inter-
calated TPP, provides one or two peaks of mass loss at >500 °C
depending on the applied synthetic protocol. Thus, in DTA curves of
the TPP/LDH hybrid materials (Fig. 5b–f), one or two exothermic
peaks, corresponding to the combustion of intercalated TPP, have
been observed, while these peaks did not exist in the DTA curve of pris-
tine NO3−/Zn–Al LDH (Fig. 5a). It is noticed that the combustion of the
pristine TPP occurs at lower temperature (480 °C, not shown here)
than in intercalated TPP. This indicates that intercalated bio-molecule
is protected by LDH layers. Based on thermogravimetric analyses, the
total amount of TPP intercalated into LDH can be calculated as ~0.6,
1.18, 0.8, 0.6, 0.87 mmol TPP/g of hybrid material and the molar ratio
TPP/Al ~0.21, 0.62, 0.28, 0.17, 0.33 for TPP-7/ZnAl(ae), TPP-7/ZnAl,
TPP-7.5/ZnAl, TPP-7.75/MgAl and TPP-8/ZnAl respectively.
Thiamine-dependent enzymes catalyze the decarboxylation of
α-ketoacids by both non-oxidative and oxidative mechanisms in
vivo (Hasson et al., 1998; Kern et al., 1997; Kluger, 1987; Kluger et
al., 1995; Konig, 1998; Malandrinos et al., 2006; Schellenberger,
1998; Schütz et al., 2003; Stamatis et al., 2007). There was some evi-
dence that TPP itself, e.g., in protein-free model systems, catalyses py-
ruvate decarboxylation (Mizuhara and Handler, 1954). Τhis ability of
thiamine in a protein-free system such as immobilized thiamine
130 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133

Fig. 3. Infrared spectra of TPP, pristine NO3−/ZnAl-LDH (a) and hybrid materials TPP-7/ZnAl-LDH(ae) (b), TPP-7/ZnAl-LDH (c), TPP-7.5/ZnAl-LDH (d), TPP-7.75/MgAl-LDH (e) and
TPP-8/ZnAl-LDH (f). On the right side: Band component analysis of the FT-IR spectrum of TPP-7/ZnAl(ae) and TPP-7/ZnAl-LDHs, in the region 450–700 cm−1.

pyrophosphate molecules on silica surface, has previously confirmed heterogeneous catalysts is tested for the decarboxylation of pyruvate.
(Vartzouma et al., 2002). The immobilized TPP showed high catalytic Pyruvate decarboxylation catalyzed by TPP adducts occurs via acetoine
activity for pyruvate decarboxylation demonstrating that it is a very formation:
active biocatalyst even more efficient than the homogeneous one
(Vartzouma et al., 2002). In order to evaluate if TPP remains intact − TPP−catalyst
2CH3 COCOO → 2CO2 þ H3 CCOCHðOHÞCH3 :
and available into LDH matrix, the reactivity of hybrid materials as pyruvate acetoine

To examine the effectiveness of homogeneous (pristine TPP) and

Table 1 heterogenized systems (TPP/LDH), a [substrate: catalyst] molar ratio
Infrared frequencies and assignments of the vibration modes of the hybrid materials [10:1] was used. The kinetic study of pyruvate decarboxylation in the
and ‘net’ TPP. presence of pristine TPP, TPP-7/ZnAl-LDH(ae) and TPP-7/ZnAl-LDH, is
Hybrid materials TPP Vibration assignments (Joshi et al., 2009; represented in Fig. 6 where the reaction yield is related to substrate con-
Karakassides et al., 2004; Leopold et al., version. Based on these catalytic data, it is observed that TPP intercalated
2005; Saranti et al., 2006; Simons, 1978; molecules into LDH exhibited comparable activity to the homogenous
Stamatis et al., 2007)
system (TPP) confirming that TPP molecules retain their bio-availability
1475, 1560, 1600 1542, 1592, Stretching of pyrimidine ring and reactivity after the intercalation procedure. The high TPP loading in
1630 cm−1 TPP-7/ZnAl-LDH catalyst, which results probably in tight packing of TPP
1655 cm−1 1650 cm−1 Bending of pyrimidine ring
into LDH layers, could explain the kinetic hysteresis observed at the be-
1685 cm−1 Stretching of C_N
477 cm −1
470 cm−1 Bending of P\O ginning of the catalytic reaction. This indicates that high load and packing
723, 773, 940, 955, 713, 763, Stretching of O\P\O, P\O, P_O of TPP could inhibit the approach of pyruvate substrate.
1060, 1224 cm−1 920, 991, Deintercalation reactions of the hybrid materials TPP-7/ZnAl-LDH(ae)
1012, 1200 cm−1
and TPP-7/ZnAl-LDH were carried out with carbonate ions, in 0.3 M
1104 cm−1 1103 cm−1 Stretching of C\O
2972 cm−1 2914, 2945, Stretching of CH (CH2\CH2) Na2CO3 aqueous solutions. The amounts of deintercalated TPP molecules,
2985 cm−1 after reaction for 24 h, were estimated by the characteristic absorption
3200 cm−1 3160 cm−1 Stretching of _C\H aromatic rings peak at 245 nm in UV spectra of TPP, using a calibration curve (Fig. 7).
3255, Stretching of Ν\Η (ΝΗ2), Ο\Η The total deintercalated amount of TPP was calculated to be 1.14 and
3385 cm−1
0.587 mmol/g for TPP-7/ZnAl-LDH and TPP-7/ZnAl-LDH(ae) respectively
 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133 131

Fig. 5. DTA curves of pristine NO3−/Zn–Al LDH (a) and hybrid LDHs TPP-7/ZnAl(ae)
(b), TPP-7/ZnAl (c), TPP-7.5/ZnAl (d), TPP-7.75/MgAl (e) and TPP-8/ZnAl (f).

Fig. 4. %TG curves of TPP, pristine NO3−/Zn–Al LDH (a) and hybrid LDHs TPP-7/ZnAl(ae)
(b), TPP-7/ZnAl (c), TPP-7.5/ZnAl (d), TPP-7.75/MgAl (e) and TPP-8/ZnAl (f).

and it is in agreement with thermogravimetric analysis. It seems that

the amount of intercalated TPP molecules into TPP-7/ZnAl-LDH is
about two times larger than the corresponding amount into the
TPP-7/ZnAl-LDH(ae); this can be explained taking into account that
the metal ratio of basal layers is Zn2+/Al3+ = 2 and 3, respectively.
Fig. 8 shows the FT-IR spectra of TPP-7/ZnAl-LDH(ae) and
TPP-7/ZnAl-LDH before and after the deintercalation reaction in
0.3 M Na2CO3 aqueous solution in comparison with the FT-IR spec-
trum of the CO32 −/Zn–Al LDH as reference sample.
Absorption bands assigned to TPP are not detected in the spectra of
deintercalated materials (Fig. 8b and d), however the appearance of
new absorption bands at 1357 and 1048 cm−1 is attributed to the vi-
Fig. 6. Time-dependent reaction profiles for pyruvate decarboxylation catalyzed by ho-
bration modes v3 and v1 of intercalated carbonate anions (Kloprogge mogenous system TPP, TPP-7/ZnAl-LDH(ae) and TPP-7/ZnAl-LDH catalysts. Conditions:
et al., 2002, 2004). These data confirm the complete replacement of The catalytic reactions were carried out at 37 °C in MeOH (1 ml) with pyruvate
TPP molecules by carbonate ions into the interlayer space of LDHs. In (200 μmol), TPP catalyst (20 μmol) and NaOH (40 μmol).
132 M. Baikousi et al. / Applied Clay Science 75–76 (2013) 126–133
Fig. 7. UV spectra of the solutions that were obtained after 24 h deintercalation reac-
tions of TPP/LDHs with carbonate ions. Inset: UV spectra of aqueous solutions of TPP
in concentration range 4.3 × 10−3–1.5 × 10−1 μmol/ml and the corresponding calibra-
tion curve.
addition, in the spectra of deintercalated materials, the absorption
bands at 555, 787 and 618 cm−1 are clearly shown. These bands are
assigned to hydroxyl translation modes of LDH's layer structure
(Kloprogge and Frost, 1999).
Fig. 9 shows the XRD pattern of TPP-7/ZnAl-LDH(ae) before and
after the deintercalation reaction in 0.3 M Na2CO3 aqueous solution
in comparison with the XRD pattern of the CO32−/Zn–Al LDH as refer-
ence sample. The higher intensity diffraction peaks at 2.52 and
1.5 nm of TPP-7/ZnAl-LDH(ae) are completely disappeared after the
deintercalation reaction; now a new diffraction peak appeared at
0.75 nm indicating the presence of carbonate ions.
Fig. 8. FT-IR spectra before and after deintercalation reactions in comparison with the FT-IR
spectra of CO32−/Zn–Al LDH as reference sample. TPP-7/ZnAl-LDH(ae) before (a) and after
(b) deintercalation. TPP-7/ZnAl-LDH before (c) and after (d).
Fig. 9. XRD patterns (a) before and (b) after deintercalation reaction of TPP-7/ZnAl-LDH(ae)
in comparison with the XRD pattern of the CO32−/Zn–Al LDH as reference sample (c).
4. Conclusion
Thiamine pyrophosphate has been successfully intercalated into
LDH by anion-exchange reactions and the co-precipitation method.
The new biomolecule/inorganic hybrids showed a significant activity
as heterogeneous catalysts and comparable one to the homogenous
TPP system for pyruvate decarboxylation. This verifies the safe stor-
age and availability of TPP into LDH. To further prove this capability
of the developed TPP/LDH material, deintercalation reaction of these
materials was performed in the presence of carbonate ions. We
found that the intercalated TPP can be easily released from the host
lattice by carbonate ions. Thus, TPP/LDH hybrids could be used as thi-
amine pyrophosphate's absorbents and reservoirs for putative usages.
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